In the last ten years there has been a remarkable renaissance in vitamin D research. Two key concepts have underpinned this renewed interest in the health benefits of vitamin D. First is the continuing debate on the worldwide prevalence of vitamin D-insufficiency, and how optimal vitamin D status can be safely achieved through conventional exposure to sunlight and dietary intake. Second is the potential for vitamin D to promote health benefits beyond its classical effects on the skeleton. Following a recent data review, the Institute of Medicine (IOM) has issued statements aimed at addressing some of the key questions concerning our new perspective on vitamin D and human health. The Recommended Dietary Allowance of vitamin D for all age groups was elevated based on bone responses to vitamin D. However, the IOM report also recognized the need for further research to better define 'non-classical' health benefits of vitamin D. The long-term impact of these recommendations is crucially dependent on one question - how does one define vitamin D-sufficiency and -insufficiency? The proposed project describes a new paradigm for quantifying optimal vitamin D and its relation to human health. The overall aim is to demonstrate that vitamin D activity is not simply defined by total serum levels of 25-hydroxyvitamin D (25D) but instead depends on the bioavailability of this metabolite to target cells and its subsequent conversion to active 1,25-dihydroxyvitamin D (1,25D) via the enzyme 1-hydroxylase (CYP27B1). The proposal hypothesizes that the ability of 25D to access target cells is influenced by its association with the serum vitamin D binding protein (DBP), with 'free' rather than 'DBP-bound' 25D being the bioactive form of this metabolite. The overall objective of the proposal will be to investigate the impact of DBP on the bioactivity of 25D using both mouse and human models. Studies using transgenic, knockout, and humanized mice will investigate how variations in the concentration and vitamin D metabolite binding affinity of DBP affect the response of these mice to 25D and 1,25D under conditions of vitamin D-sufficiency and -deficiency. Data from these experiments will then be related to studies in humans, where DBP concentration and binding affinity are strongly influenced by genetic variations in the DBP gene. Human studies will incorporate analysis of DBP and free 25D/1,25D in a large patient cohort with multiple measures of vitamin D function, but will also involve a pilot supplementation study utilizing parental vitamin D or 25D. These analyses will employ a new mathematical algorithm for determining serum free 25D and 1,25D and will use novel assay technology to physically measure serum levels of free 25D. This model not only puts forward a new paradigm for defining optimal vitamin D status but also aims to highlight a more 'personalized' perspective on vitamin D health that will incorporate both classical and non-classical actions of vitamin D. .